Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+.
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ABSTRACT:
SUBMITTER: Irvine RF
PROVIDER: S-EPMC1147508 | biostudies-other | 1986 Dec
REPOSITORIES: biostudies-other
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Project description:Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.
| S-EPMC2084373 | biostudies-literature
Project description:We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.
| S-EPMC1149131 | biostudies-other
Project description:Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues were synthesized and their effects on [3H]Ins(1,3,4,5)P4 5-phosphatase, [3H]Ins(1,3,4,5)P4 3-phosphatase and [3H]inositol 1,4,5-trisphosphate [3H]Ins(1,4,5)P3] 5-phosphatase activities were examined. The Ins(1,3,4,5)P4 analogue with the aminobenzoyl group at the 2-position of Ins(1,3,4,5)P4 inhibited the hydrolysis of 5-phosphate of [3H]Ins(1,3,4,5)P4 catalysed by erythrocyte ghosts, with a lower Ki value than seen with Ins(1,3,4,5)P4, whereas the analogue with the aminocyclohexanecarbonyl group at the same position had a higher Ki value. The Ins(1,4,5)P3 analogues that we had previously synthesized were also capable of inhibiting this process, with the same tendency as Ins(1,3,4,5)P4 analogues. Such differences in the potency among Ins(1,3,4,5)P4 and Ins(1,4,5)P3 analogues were applicable to other phosphatase activities, namely [3H]Ins(1,3,4,5)P4 3-phosphatase and [3H]Ins(1,4,5)P3 5-phosphatase. These results suggest that the active sites of these enzymes may catalyse the dephosphorylation in a similar fashion.
| S-EPMC1151095 | biostudies-other
Project description:We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3-sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3-stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor.
| S-EPMC1217129 | biostudies-other
Project description:The influence of highly phosphorylated inositol phosphates on the Ins(1,3,4,5)P4 3-phosphatase enriched from the soluble fraction of pig brain was tested, using [5-32P]Ins(1,3,4,5)P4 as substrate. Both Ins(1,3,4,5,6)P5 and InsP6 were very potent inhibitors of the Ins(1,3,4,5)P4 3-phosphatase. The Ki values were approximately 60 nM and approximately 3 nM for Ins(1,3,4,5,6)P5 and InsP6 respectively. Ins(1,3,4,5,6)P5 and InsP6 also inhibited the Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. Using Ins(1,3,4,5)P4 as substrate, the Ki values were about 35 microM and 15 microM for Ins(1,3,4,5,6)P5 and InsP6 respectively. The concentrations which led to a 50% inhibition of Ins(1,4,5)P3 (0.5 microM) degradation by the 5-phosphatase were about 20 and 10 microM for the pentakis- and hexakis-phosphate respectively. As the intracellular concentrations of Ins(1,3,4,5,6)P5 and InsP6 are high (up to 60 microM) compared with those of the inositol trisphosphates and tetrakisphosphates, it is possible that the highly phosphorylated inositol phosphates act as regulators in the metabolism of Ca(2+)-mobilizing inositol phosphates.
| S-EPMC1151471 | biostudies-other
Project description:Following our studies on the identification of a calsequestrin-like protein (CSLP) from sea-urchin eggs [Oberdorf, Lebeche, Head & Kaminer (1988) J. Biol Chem. 263, 6806-6809], we have characterized its Ca(2+)-binding properties and identified it as a glycoprotein. The molecule binds 23 mol of Ca2+/mol of protein, as determined by equilibrium dialysis. This is in the range reported for cardiac calsequestrin but is about half the binding capacity of striated muscle calsequestrin. The affinities of the CSLP for Ca2+ are decreased by increasing KCl concentrations (20-250 mM) and the presence of Mg2+ (3 mM) in the medium: the half-maximal binding values varied from 1.62 to 5.77 mM. Hill coefficients indicated mild co-operativity in the Ca2+ binding. Ca2+ (1-8 mM)-induced u.v. difference spectra and intrinsic fluorescence changes suggest a net exposure of aromatic residues to an aqueous environment. C.d. measurements showed minor Ca(2+)-induced changes in alpha-helical and beta-sheet content of less than 10%. These spectral changes are distinctly different from those found in muscle calsequestrin. Immunoblotting studies showed that the CSLP is distinct from calreticulin, a low-affinity Ca(2+)-binding protein.
| S-EPMC1133070 | biostudies-other
Project description:1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.
| S-EPMC1150196 | biostudies-other
Project description:Ins(1,3,4,5)P4 has been suggested to be involved in cellular Ca2+ signalling. A receptor from pig cerebellar membranes, which binds InsP4 with high affinity and selectivity [Donié & Reiser (1989) FEBS Lett. 254, 155-158], has been solubilized and purified about 20,000-fold by chromatography using CM-cellulose, heparin-agarose and hydroxyapatite. The InsP4 receptor, identified by SDS/PAGE, had an apparent molecular mass of 42 kDa and bound 4.6 nmol of InsP4 per mg of protein, with a dissociation constant of 5.6 nM.
| S-EPMC1150074 | biostudies-other
Project description:In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.
| S-EPMC3133919 | biostudies-literature
Project description:Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.
| S-EPMC1136905 | biostudies-other